title: Preparation of protein samples for NMR structure, function, and small-molecule screening studies.
abstract: In this chapter, we concentrate on the production of high-quality protein samples for nuclear magnetic resonance (NMR) studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium and outline our high-throughput strategies for producing high-quality protein samples for NMR studies. Our strategy is based on the cloning, expression, and purification of 6×-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6×-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (>97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this chapter describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening, and structural genomics research.
authors.affiliation: Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Acton
authors.forename: Rong
authors.initials: R
authors.lastname: Xiao
authors.forename: Stephen
authors.initials: S
authors.lastname: Anderson
authors.forename: James
authors.initials: J
authors.lastname: Aramini
authors.forename: William A
authors.initials: WA
authors.lastname: Buchwald
authors.forename: Colleen
authors.initials: C
authors.lastname: Ciccosanti
authors.forename: Ken
authors.initials: K
authors.lastname: Conover
authors.forename: John
authors.initials: J
authors.lastname: Everett
authors.forename: Keith
authors.initials: K
authors.lastname: Hamilton
authors.forename: Yuanpeng Janet
authors.initials: YJ
authors.lastname: Huang
authors.forename: Haleema
authors.initials: H
authors.lastname: Janjua
authors.forename: Gregory
authors.initials: G
authors.lastname: Kornhaber
authors.forename: Jessica
authors.initials: J
authors.lastname: Lau
authors.forename: Dong Yup
authors.initials: DY
authors.lastname: Lee
authors.forename: Gaohua
authors.initials: G
authors.lastname: Liu
authors.forename: Melissa
authors.initials: M
authors.lastname: Maglaqui
authors.forename: Lichung
authors.initials: L
authors.lastname: Ma
authors.forename: Lei
authors.initials: L
authors.lastname: Mao
authors.forename: Dayaban
authors.initials: D
authors.lastname: Patel
authors.forename: Paolo
authors.initials: P
authors.lastname: Rossi
authors.forename: Seema
authors.initials: S
authors.lastname: Sahdev
authors.forename: Ritu
authors.initials: R
authors.lastname: Shastry
authors.forename: G V T
authors.initials: GV
authors.lastname: Swapna
authors.forename: Yeufeng
authors.initials: Y
authors.lastname: Tang
authors.forename: Saichiu
authors.initials: S
authors.lastname: Tong
authors.forename: Dongyan
authors.initials: D
authors.lastname: Wang
authors.forename: Huang
authors.initials: H
authors.lastname: Wang
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
mesh_terms: D003001:Cloning, Molecular; D019295:Computational Biology; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis; D005285:Fermentation; D023281:Genomics / Q000379:methods; D007553:Isotope Labeling; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010940:Plant Proteins / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D054852:Small Molecule Libraries / Q000302:isolation & purification; D014908:Triticum / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D010940:Plant Proteins; D011506:Proteins; D054852:Small Molecule Libraries